Real-time monitoring apparatus for biochemical reaction

ABSTRACT

The present invention relates to an apparatus for real-time monitoring chemical reaction between various biomaterials. More particularly, the present invention directed to a real-time monitoring apparatus for biochemical reaction, which comprises parabolic mirror and/or an optical waveguide tube for effective irradiation of light over the whole plate with uniform intensity.

TECHNICAL FIELDS

The present invention relates to an apparatus for real-time monitoringchemical reaction between various biomaterials. More particularly, thepresent invention is directed to a real-time monitoring apparatus forbiochemical reaction, which comprises a temperature control block systemcomprising a thermoelectric element, capable of supplying heat into areaction tube, a heat transmission block which transmit the heat to thereaction tubes; a light irradiation source part comprising a lamp whichirradiates light with uniform intensity to sample contained in thereaction tube, and optical waveguide; and optical system comprising alight receiving part for receiving fluorescence irradiated from thesample by the light emitted from the light irradiation source.

BACKGROUND ART

The present invention relates to an apparatus for real-time monitoringchemical reaction between various biomaterials. More particularly, thepresent invention is directed to a real-time monitoring apparatus forbiochemical reaction, which comprises a temperature control block systemcomprising a thermoelectric element, capable of supplying heat into areaction tube, a heat transmission block which transmit the heat to thereaction tube; a light irradiation source part comprising a lamp whichirradiates light with uniform intensity on sample contained in thereaction tube, and optical waveguide; and optical system comprising alight receiving part for receiving fluorescence irradiated from thesample by the light emitted from the light irradiation source.

Recently, the research and development for chemical microprocessor havebeen performed actively, which can carry out pretreatment of sample,reaction, separation, detection and etc., within a single chip, socalled lab-on-a-chip. The lab-on-a-chip composed from glass, silicon orplastic material and manufactured through the lithography technologywhich has been employed for semiconductor chip, mounts micro-sizeddevice for analyzing samples rapidly and sensitively.

The above all procedures required for the analysis of sample, i.e.,pretreatment, reaction, separation, detection and etc., can be performedcontinuously. In addition, by using the chip the time needed for thesample analysis can be reduced to second or minute level and also theamount of sample can be reduced to micro-liter level and the size ofapparatus on which the chip is mounted can also be miniaturized. Thelab-on-a-chip technology is based on the capillary electrophoresisdeveloped by Harrison in early 1990s, and had been started to be knownto the public by the fact that a small size lab. Device for thecapillary electrophoresis analysis can be integrated into a single chip.

Meanwhile, recently, so called real-time PCR technology which can checkrapidly the progress of every cycle of amplification reaction bydetection of fluorescence from reaction tubes, without using theseparation step in gel phase. The conventional apparatus employed forthis real-time PCR technology may be manufactured by coming the thermalcycler for PCR and the fluorometer for detection of products.

The conventional real-time PCR apparatus is composed of thethermoelectric element, the heat transmitting block which transmit heatinto reaction tubes which contains sample, the light irradiation sourcewhich irradiate light into sample contained in the reaction tube, andthe light receiving part which accept the fluorescence generated fromthe sample.

By using the conventional real-time PCR apparatus, the progress of PCRcan be checked in real-time by measuring the intensity of fluorescencegenerated from sample upon completion of each cycle by the operation ofthe thermoelectric element for cooling and heating repeatedly to carryout the reaction of biochemical sample contained in the tube.

In the conventional real-time PCR apparatus, in general, a halogen lampand a metallic halide lamp have been employed as a light irradiationsource. In this conventional apparatus, a selective transmissionfilter(9) transmit selectively the light with preferred wavelength fromthe light radiated from the lamp(5) and then, the light thus selectedirradiated into the sample contained in the tubes via a reflectionmirror(18) and through a condensing lens(17).

Then, the sample contained in the tubes generate fluorescence byirradiation of light thus selected. The fluorescence thus generated, isreflected by reflection mirror(18) and focused through condensinglens(17). The fluorescence thus focused from each tubes is imaged on alight receiving element of the light receiving part to indicate andrecord the progress of reaction, continuously.

By the way, the light intensity generated from the light radiation lampof the conventional apparatus, is differentiated depends on thepositions, i.e., the center or the edges of the facet of light beam.Consequently, the light intensity generally varied with gaussian curvealong with central axis of the facet of light beam and maintain thisdistribution even through the condensing lens.

This difference of brightness of light between the center and the edgesof the facet of light beam, may cause a problem that the accurate datacannot be obtained since the fluorescence response from the samplepositioned at the edges is weak. Various study and development have beentried to obviate this problem of the prior art. However, the technologywhich can overcome the above mentioned problem has not yet beensuggested up to now. That is, a technology which can irradiate lightwith uniform intensity on the whole area of tube plate enlarged day byday, has not known in this field.

For the above reason, there is a critical limitation for enlarging thenumber of the tubes which can be monitored simultaneously. Therefore,the primary object of the present invention is to provide an apparatusfor real-time monitoring the progress of biochemical reaction, whichhave technical part to irradiate light with uniform intensity over thewhole area of the tube plate which is enlarged than that of prior art.

In addition, as depicted in FIG. 5, the reaction tube plate of prior artis in general rectangular shape. However the conventional lightradiation system composed of a lamp and a lens, emits plane wave beam ofwhich facet has round shape. Thus, some edge part of light beam shouldbe eliminated to be fitted with the rectangular shaped tube plate. Thisproblem lowers the efficiency of the source due to eliminated pat oflight beam.

Various studies to obviate this problem of the low efficiency of lightsource have been suggested. However, any appropriate tools which can beadapted to a laboratory device integrated compactly, have not yet beendeveloped.

Therefore, the present inventors had conceived that the sensitivity ofthe monitoring apparatus can be improved by strengthening the intensityof light by providing the light beam through waveguide which has asimilar facet shape as that of the tube plate. Thus, another object ofthe claimed subject matter is to provide a real time monitoringapparatus for biochemical reaction which can irradiate light withuniform intensity over the whole area of the reaction tube plate, evenover the whole area of the reaction tube plate enlarged than that ofprior art, by irradiating light beam through a light irradiation partvia a mirror.

BRIEF DESCRIPTION OF THE DRAWINGS

The above objects and other advantages of the present invention willbecome more apparent by describing in detail a preferred embodimentthereof with reference to the attached drawings, in which:

FIG. 1 is a schematic diagram of a real-time monitoring apparatus of thepresent invention for checking the process of biochemical reactionbetween various biological samples.

FIG. 2 is a schematic diagram of an apparatus of prior art for real-timemonitoring of the progress of biochemical reaction.

FIG. 3 represents the distribution of the luminosity on the tube plateof the real-time monitoring apparatus of the present invention. HereinFIG. 3 a shows a tube plate and FIG. 3 b shows the light intensitydistribution in X direction and in Y direction.

FIG. 4 is a schematic diagram of the optical waveguide for plane wavelight in real-time monitoring apparatus of the present invention. HereinFIG. 4 a shows a waveguide. FIG. 4 b shows the distribution curve of thelight intensity. FIG. 4 c shows a schematic diagram of total internalrefraction.

FIG. 5 is a schematic diagram of radiation system for plane wave lightin the real-time monitoring apparatus of prior arts.

FIG. 6 is a schematic diagram of radiation system for plane wave lightin real-time monitoring apparatus of the present invention.

FIG. 7 is another schematic diagram of light radiation system for planewave light in real-time monitoring apparatus of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS MARK

-   1: heat sink plate-   2: thermoelectric element-   3: heat transmission block-   4: reaction tube-   5: lamp-   6: Infra-Red cutting filter-   7: reflective mirror 1-   8: optical waveguide-   9: selective transmission filter 1-   10: focusing lens 1-   11: reflective mirror 2-   12: light received element-   13: focusing lens 2-   14: selective transmission filter 2-   17: condensing lens-   18: reflective mirror 3-   25: incident light-   26: the emitting light-   28: distribution of the light intensity-   33: condensing lens 2-   34: tube plate-   36: focusing lens 3-   38: condensing lens 3-   40: lamp with ellipsoidal mirror-   41: lamp with parabolic mirror

DISCLOSURE OF THE INVENTION

The object of the present invention it to provide a real-time monitoringapparatus for biochemical reaction comprising: a temperature controlblock comprising a thermoelectric element(2) capable of supplying theheat into a reaction tube(4) and a heat transmission block(3) whichtransmit the heat to the reaction tube(4) containing a sample; a lightirradiation source part comprising a lamp(5) which irradiates the lightwith uniform intensity of a sample contained in the reaction tube, aInfra-Red cutting filter(6) cutting Infra-Red from the lamp, an opticalwaveguide(8) and a first focusing lens(10) for obtaining uniform lightbeam of the light from the lamp in a wide area, a selective transmissionfilter 1(9) for transmitting light selectively to monitor a reactionprogress selectively, a second reflective mirror(11), and firstcondensing lens(17); an optical system comprising a light receivingpart(12, 13 and 14) for receiving fluorescence(15) generated by thelight emitted from the light irradiation source.

Furthermore, another object of the present invention is to provide areal-time monitoring apparatus for biochemical reaction which has aellipsoidal mirror to get an uniform light intensity distribution.

The light from the light radiation lamp (5) is focused into the lightwaveguide (8). The light in the light waveguide is propagated in amanner of total internal reflection. The light beam at the end of thelight waveguide (8) become a uniform 2-dimensional light source and arefocused on the samples contained in the reaction tubes (4) through thefirst condensing lens (10). By using the uniform light beam (31), therereaction progress may be more easily measured over the whole range ofthe reaction tubes.

Therefore, an apparatus capable of checking the progress of reactionmore efficiently may be provided due to the reduced variation ofradiation light intensity in each reaction tube by irradiating the lightwith uniform intensity over broader reaction tube area. The apparatusenables to process uniform reaction information while reacting varioussamples simultaneously in the reaction tubes.

Still another object of the present invention is to provide a real-timemonitoring apparatus for biochemical reaction with improved reactionmonitoring sensitivity. In the improved apparatus of the presentinvention, the usage efficiency of light source is improved byincreasing the amount of light irradiated into the reaction tubes, asthe result of minimizing the loss of the light source in prior art byirradiating rectangular light beam(37) adjusted to the aspect ratio ofthe rectangular reaction tube plate using a light waveguide(8).

The present invention relates to a measuring and monitoring apparatusfor reaction progress in real-time while reacting various samples. Moreparticularly, the present invention directed to a real-time monitoringapparatus for biochemical reaction comprising: a temperature controlblock comprising a thermoelectric element capable of supplying heat intoreaction tubes and a heat transmission block for transmitting heat tothe reaction tubes containing samples; a light irradiation sourcecomprising a lamp which irradiates light with uniform intensity on thesamples contained in the reaction tubes, a condensing lens and anoptical waveguide; an optical system comprising a light receiving partfor receiving fluorescence from the samples contained in the reactiontubes generated by the light irradiated from the light irradiationsource.

The temperature control block of the present invention is comprised ofthermoelectric element(2) for repeating cooling and heating cycles andheat transmission block(3) for transmitting heat to reaction tubescontaining samples. Moreover, a radiating plate(1) may be additionallyprovided for increasing the efficiency of the thermoelectric element.When the cooling and heating process is carried out repeatedly by thetemperature control block, the amount of biological samples in thereaction tubes in terms of a genetic amplification reaction will begradually amplified to 2^(n) (n: the number of repeating cycle).

In the real-time monitoring apparatus for biochemical reaction of thepresent invention, the optical system comprised of a light irradiationsource and a light receiving part for measuring the amplified reactionprogress in real-time is placed on a temperature control block.

The light irradiation source of the present invention comprises alamp(5) which irradiates light to a sample contained in reaction tubes,a Infra-Red cutting filter(6) intercepting the light from the lamp, anoptical waveguide(8) and a first focusing lens (10) for obtaininguniform light beam of the light from the lamp in a wide area, aselective transmission filter 1(9) for transmitting light of specificwavelength selectively to monitor a reaction progress in the tube,condensing lens 1(17) for receiving the fluorescence(15) by theradiation light(16) and a second reflective mirror(11) which alterslight path.

In the prior art, it is a fundamental problem that light intensity inthe center of a reaction tube plate and light intensity(32) at the edgesof the reaction tube plate are different each other. The presentinvention employs an optical waveguide(8) placed in front of the lightsource to reduce of the difference in light intensity between the centerand the edges of the tube plate and solves the problem of the prior art.

The waveguide(8) is designed to irradiate the light beam from the lightsource uniformly over a wider range. The light beam from the lightsource experience a total internal reflection due to the refractiveindexes between a propagating medium(n2) in the waveguide(8) andsurrounding air(n1). The light beam entered into the waveguide(8) formsa uniform plane wave light source at the light outlet(27) thereof.

Generally, in the lamp or lens-type optical system, light intensity ismore densely distributed in the center and less densely distributed atthe edges. The light intensity at the edges is merely about 50-60% ofthat in the center. Due to the difference in light intensity, samplesplaced in the center shows a higher sensitivity and reaction level thanthe samples placed at the edges by the different amount of lightintensity there between.

In prior art to solve the above problem, analysis has been made byadjusting the measured light intensity to the light intensity at theedges. This brings the sensitivity of the whole apparatus degraded. Inaddition, in case of using a ultra-sensitivity light receiving elementto overcome the degradation problem of sensitivity, the apparatusrequires the increase in size and costs.

The present invention solves the prior art problem by forming a firstreflecting mirror(7) of the radiation light source (5) as a ellipsoidalmirror. With this structure, light beam are focused at one point and areput into the optical waveguide(8) as many as possible. The opticalwaveguide(8) is designated to provide light intensity more uniformlyover the broader cross-section of light beam. The waveguide(8) employsthe difference in refractive indexes between the propagation medium(n2)and surrounding air (n1). Light beam where an incident angle(i) of theincident beam(29) has equal to or larger than a critical angle(c)experience total internal reflection(30) inside the waveguide(8). Theincident beam(29) are emitted from the light outlet(27) through thewaveguide(8) without loss in light intensity due to the lightdistribution. In this manner, a highly uniform 2-diemnsional lightsource is formed at the light outlet(27) of the waveguide(8).

In the present invention, the refractive index of the propagation mediumin the optical waveguide(8) is preferably 1.35˜2.0. The total internalreflection condition in a medium should satisfy that an incidentangle(i) of incident light beam(29) is equal to or bigger than acritical angle(c). In FIG. 4, reference numeral 30 represents thereflected light beam, 26 for the emitting light beam, and 32 for therefracted light.

Under the condition of n1Sin(i)=n2Sin(o),

-   -   n1=1.0, sin(o)=1 (n1: refractive index of air, o=90 degree)    -   sin(c)=n2 (i (incident angle)>c (critical angle)

As shown in FIG. 3, a plane wave light source of the present inventionwith uniformly distributed intensity over the cross-section of lightbeam has more than 85% (21) of light intensity at the edges comparedwith the light intensity in the center of reaction tube plate(34) andenables to monitor the reaction progress more uniformly by achievingsubstantially improved light intensity uniformity compared with theprior art.

On the other hand, the typical reaction tube plate(34) used forproceeding the reactions of the various biological samplessimultaneously is usually a rectangular shape, while the prior artoptical radiation system comprising a lamp and a lens generates planewave light beam with a round-type cross-section.

Therefore, as shown in FIG. 5, plane wave light beam with a round-typecross-section are adjusted to the shape of the rectangular reaction tubeplate(34) and the remaining portions(35) unnecessary for the rectangularshape are removed. The removal of light beam(35) results to the removalof a portions of light beam from the light source lamp and causes gradedefficiency of the light source. In FIG. 5, reference numeral 35represents the removed light beam.

However, as shown in FIG. 6, the light waveguide(8) of the presentinvention has a shape of a rectangular cross-section(37) to be fittedwith the aspect ratio of the rectangular plate(34) so that it is able touse the light from the lamp in it maximum efficiency because there areno light beam to be removed. As referenced by the reference numerals 21and 24 in FIG. 3, the monitoring sensitivity of present invention isimproved by increasing the light intensity irradiated on the plate tomore than 20%. In FIG. 3, the reference numerals 21 and 24 represent thebrightness distribution along the X-axis and Y-axis, respectively.Reference numerals 22 and 23 represent the brightness distribution alongthe X-axis and Y-axis, respectively, of prior art.

The radiation light controlled to a plane wave light source shape isirradiated on the samples contained in the reaction tubes(4) placed onthe plate through a first focusing lens(10), a first selectivetransmission filter(9), a second reflecting mirror(11) and a firstcondensing lens. The nucleic acid samples in the reaction tubes areamplified per every cycle by the temperature block and fluorescence isgenerated from the amplified samples by the radiation light.

Meanwhile, the light receiving part of the present invention comprises asecond focusing mirror(13) imaging fluorescence(15) from the samplescontained in the reaction tubes(4) to the direction of an imagingelement by the first condensing lens(17) and the second reflectingmirror(11); a light receiving element(12) for recoding the imaged figureby the second focusing lens(13) and a second selective transmissionfilter(14).

The fluorescence(15) generated from the samples is imaged on the lightreceiving element(12) by a second focusing lens(13) through thecondensing lens 1(17), the reflecting mirror 2(11) and the secondselective transmission filter (14). The images by fluorescence of eachsample are transferred to a computer which analyzes the changingreaction progress per every cycle. Furthermore, it is possible tocompare and analyze while per each sample by reacting variousbiochemical samples in the reaction tube plate described analyzing thereaction progress for each sample.

As described above, the real-time monitoring apparatus for biochemicalreaction of the present invention, employs the lamp(S) with theellipsoidal-type mirror and the optical waveguide(8), and there may becapable of monitoring the biological reaction progress with uniformsensitivity over the whole reaction plate area over the whole reactionplate(34).

Using the apparatus of the present invention with uniform lightintensity, the present invention efficiently performs the comparativeanalysis of various samples while reacting the various samplessimultaneously on one reaction tube plate by monitoring the reactionprogress of the samples contained in the reaction tubes between thecenter and the edges of the reaction tube plate, more accurately.

According to the prior art, the reaction progress of the samplescontained in the reaction tubes is equal to in the center and at theedges while there are limitations when performing a comparative analysisof the reaction progress for the samples due to the difference in lightintensity of measured fluorescence. The present invention overcomes thelimitation of prior art. Therefore, the real-time monitoring apparatusfor biochemical reaction of the present invention is able to provide anappropriate apparatus for performing a comparative analysis of thereaction progress of the various samples by minimizing variation oflight detection sensitivity during the reaction in the reaction tubeplate.

Moreover, the present invention provides a profile of the light beamfrom the light source which is adjusted to a rectangular shape to befitted with the aspect ration of the rectangular shape of the reactiontube plate. This adjusted profile of the present invention excludes thenecessity of removing some portions of the light beam in the prior artand enables to use the light beam from the light source lamp at themaximum efficiency which improves energy efficiency of the presentapparatus accordingly.

What is claimed is:
 1. A real-time monitoring apparatus for biochemicalreaction, comprising: a thermoelectric element capable of supplying heatinto reaction tubes; a heat transmission block which transmits the heatto the reaction tubes; a tube plate capable of holding a sample; a lampfor irradiating to a sample contained in at least one of the reactiontubes in the tube plate; at least one reflective mirror; an opticalwaveguide positioned in front of the lamp which has an open structure incooperation with said reflective mirror, said optical waveguide having aconfiguration that alters a light path passing through at least one endof the optical waveguide and provides a uniform intensity of light; aninfra-red cutting filter filtering light transmitted through a lightpath that comprises said infra-red cutting filter, said reflectivemirror and the optical waveguide and said infra-red cutting filtercutting infra-red from the lamp and a selective transmission filter fortransmitting light selectively to monitor a reaction progress; saidlight transmitted through a light path illuminating the sample with auniform light intensity distribution as provided by the uniformintensity of light from the optical waveguide, the optical waveguidereducing of the difference in light intensity between the center and theedges of the tube plate; a condensing lens positioned outside of aportion of a light path comprising said reflective mirror, the opticalwaveguide and the infra-red cutting filter; an optical system comprisinga receiving part for receiving fluorescence transmitted through a secondfocusing lens, using a light receiving element capable of receiving thefluorescence, the fluorescence irradiated from the sample by lightemitted from a light irradiation source as transmitted through a lightpath comprising the optical waveguide, the selective transmission filterand a first focusing lens; and said components arranged so that lightwill travel through optical components of the real time monitoringapparatus in the order of the lamp, the infra-red cutting filter, theoptical waveguide, the selective transmission filter, the first focusinglens through the sample and the light receiving element.
 2. Thereal-time monitoring apparatus according to claim 1, wherein the lampincludes an ellipsoidal reflecting mirror or a parabolic mirror.
 3. Thereal-time monitoring apparatus according to claim 1, wherein therefractive index of medium of the optical waveguide is 1.35˜2.0.
 4. Thereal-time monitoring apparatus according to claim 1, wherein: thereaction tube plate has a rectangular layout; and the optical waveguidehas a rectangular shape, thereby minimizing the loss of the lightemitted from the light irradiation source by irradiating the lighttransmitted through the light path in a rectangular light beam adjustedto an aspect ratio of the reaction tube plate using the opticalwaveguide.
 5. The real-time monitoring apparatus according to claim 1,wherein the cross-section of the optical waveguide has a round shape. 6.The real-time monitoring apparatus according to claim 1, furthercomprising two or more reflective mirrors positioned to alter light pathafter transmission from the light irradiation source.
 7. The real-timemonitoring apparatus according to claim 2, wherein the lamp including aparabolic mirror further comprises the first focusing lens.
 8. Areal-time monitoring apparatus for biochemical reaction, comprising: athermoelectric element capable of supplying heat into reaction tubes; aheat transmission block which transmits the heat to the reaction tubes;a tube plate capable of holding a sample; a lamp for irradiating to asample contained in at least one of the reaction tubes in the tubeplate; at least one reflective mirror; an optical waveguide positionedin front of the lamp which has an open structure in cooperation withsaid reflective mirror, said optical waveguide having a configurationthat alters a light path passing through at least one end of the opticalwaveguide and provides a uniform intensity of light; an infra-redcutting filter filtering light transmitted through a light path thatcomprises said infra-red cutting filter, said reflective mirror and theoptical waveguide and said infra-red cutting filter cutting infra-redfrom the lamp and a selective transmission filter for transmitting lightselectively to monitor a reaction progress; said light transmittedthrough a light path illuminating the sample with a uniform lightintensity distribution as provided by the uniform intensity of lightfrom the optical waveguide, the optical waveguide reducing of thedifference in light intensity between the center and the edges of thetube plate; a condensing lens positioned outside of a portion of a lightpath comprising said reflective mirror, the optical waveguide and theinfra-red cutting filter; an optical system comprising a receiving partfor receiving fluorescence transmitted through a second focusing lens,using a light receiving element capable of receiving the fluorescence,the fluorescence irradiated from the sample by light emitted from alight irradiation source as transmitted through a light path comprisingthe optical waveguide, the selective transmission filter and a firstfocusing lens; and said components arranged so that light will travelthrough optical components of the real time monitoring apparatus in theorder of the lamp, the infra-red cutting filter, the selectivetransmission filter, the optical waveguide, the first focusing lensthrough the sample and the light receiving element.